Current processes that have been developed for the manufacture of cellular products for cellular therapies, as well as ex vivo genetic manipulation of these cells, have until now been carried out manually or at best by using semi-automated procedures.
For example, cellular therapy of hereditary monogenic genetic disorders, like primary immunodeficiencies, can effectively be treated by overexpression of the wild-type gene product via viral modification of isolated CD34+ progenitor cells (Rivat et al. 2012, Human Gene Therapy 23: 668-675). The generation of this type of cellular product involves the following distinct steps (e.g. Scaramuzza et al. 2013, Mol. Ther 21: 174-184):                (1) Generation of peripheral blood mononuclear cells (PBMC) by Ficoll gradient separation or erythrocyte reduction        (2) Magnetic labeling and magnetic enrichment of the CD34+ cell population        (3) Cultivation and pre-activation of the enriched CD34+ cells with a cytokine cocktail (hTPO, hSCF, hFlt-3L, hIL-3)        (4) Transduction of the pre-activated CD34+ cells with retroviral or lentiviral vectors containing the therapeutic expression cassette        (5) Washing of the final cell product and resuspension in buffer for infusion.        
PBMC preparation, magnetic labeling and wash steps are performed manually by centrifugation in tubes and bags. CD34+ cell cultivation, pre-activation and lentiviral transduction are performed in flasks or bags coated with a fibronectin fragment with manual addition of reagents. In current optimized protocols (e.g. Scaramuzza et al. 2013, Mol. Ther.), two transduction cycles with high dose vector are performed.
If cellular therapies and gene therapies are to move from their current translational setting into routine clinical use, a standardized production of cellular therapeutic agents and their genetic modification is required.
Furthermore, genetic modification of stem cells using viral vectors is hampered by the low efficiency of viral transduction and the low numbers of stem cells available for modification from some patient groups (e.g. infants with primary immunodeficiencies). To improve transduction efficiency of stem cells, a method to cultivate the cells at enhanced concentration with a high concentration of viral vector is required (Chuck et al., 1996, Human Gene Therapy; Haas et al., 2000, Mol. Ther.). To prepare stem cells from patient samples, large volumes of blood products and large cell numbers must be processed, requiring a processing chamber with a large volumetric capacity. For genetic modification of small numbers of stem cells, ideally a small cell cultivation chamber would be used to allow cultivation at increased cell density and virus concentration.
Accordingly, it was an object of the invention to provide a process for genetic modification of stem cells starting with a cell sample having a large volume using a centrifugation chamber providing an appropriate processing volume for the cell sample and inducing genetic modification of stem cells suspended in a much smaller volume of liquid with good yield in the same (large volume) centrifugation chamber. Furthermore, the process should enable a closed and highly automated manufacturing procedure.